Staphylococcal protein A (SpA) is a major virulence factor of the important human bacterial pathogen, Staphylococcus aureus. It possesses a surprisingly large range of disparate functions, primarily through its interaction with elements of host immune and inflammatory response systems. The aims of this proposal seek to define the structural and mechanistic origins of these activities with the ultimate goal of therapeutic intervention. The emergence of community acquired S. aureus infections resistant to traditional and last-resort antibiotics is a significant and rising threat to human health. Many studies comparing wild-type strains to those lacking SpA have established that this cell surface protein plays an important role in the bacterium's ability to evade the immune system and contribute to the inflammatory sepsis that is the ultimate cause of patient death. We propose to study both of these activities by determining the structures of SpA and/or its domains in complex with antibody fragments (Fc) by X-ray crystallography. Previous studies have established that the functional half of the protein is highly flexible and consists of five nearly identical domains tha each possesses the ability to form the complexes described above. We have developed a sophisticated method to describe the interdomain orientational distributions (IOD) based on residual dipolar coupling measurements in two-domain constructs in which one domain is aligned with the magnetic field via a lanthanide binding tag. We plan to compare the IOD in the presence and absence of monomeric Fc to better understand the interdependence of the IOD and antibody binding. We also plan to construct a rigid version of SpA and various multidomain constructs by removing the C- terminal cap of helix 3 and the N-terminal cap of helix 1 of the adjacent domain. If we are successful in producing such a rigid protein, we will study the effect of rigidification on function, including antibody binding and cell surface recepto activation. To serve its various functions, the protein must be translocated through the cell membrane and attached to the peptidoglycan framework of the cell wall. We propose to study the mechanism of SpA secretion, particularly the role of the rapid unfolding/refolding (RUF) property of the N-terminal half of the protein. A better understanding of SpA secretion would allow the development of therapeutic approaches to blocking secretion, thereby eliminating all of its virulent functions. Taken together, the proposed studies offer a broad range of biophysical insights into the structure and function of a key weapon used by S. aureus to make it one of the two or three most significant bacterial threats to human health in the US. To obtain these insights, we plan to develop several new biophysical methods that will have applications to other flexible and RUF proteins.